Virulence of PathogenicCoxiella burnetiiStrains After Growth in the Absence of Host Cells

Abstract

Coxiella burnetii is a gram-negative bacterium that causes the zoonotic disease Q fever. Traditionally considered an obligate intracellular agent, the requirement to be grown in tissue culture cells, embryonated eggs, or animal hosts has made it difficult to isolate strains and perform genetic studies on C. burnetii. However, it was recently demonstrated that the attenuated Nine Mile Phase 2 (NM2) C. burnetii strain will grow axenically in acidified citrate cysteine medium (ACCM) in a 2.5% oxygen environment. The current study was undertaken to determine whether more virulent C. burnetii strains could be grown in ACCM, and whether virulence would be maintained after passage. The ACCM medium supported an ?1000-fold expansion of Nine Mile Phase 1 (NM1), NM2, M44, and Henzerling strains of C. burnetii, whereas the Priscilla (Q177) strain expanded only 100-fold, and the K strain (Q154) grew poorly in ACCM. To determine if passage in ACCM would maintain the virulence of C. burnetii, the NM1 strain was grown for up to 26 weekly passages in ACCM. C. burnetii maintained in ACCM for 5 or 8 passages maintained full virulence in a mouse model, but NM1 passaged for 23 or 26 times was somewhat attenuated. These data demonstrate that virulent strains of C. burnetii can be successfully passaged in ACCM; however, some strains can lose virulence after extended passage, and other strains grow poorly in this medium. The loss of virulence in axenic culture was associated with some truncation of lipopolysaccharide chains, suggesting a possible mechanism for attenuation.

Genotyping and Axenic Growth ofCoxiella burnetiiIsolates Found in the United States Environment

“…Starting concentrations in the cultures were evaluated by PCR targeting com1 and kept between 5 · 10 5 and 6 · 10 6 genome equivalents per mL to minimize the impact of starting density on differences in growth (Kersh et al 2011). Four separate flasks with starting densities in the range described were created for each isolate.…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Coxiella isolated from natural sources and infections produces full-length lipopolysaccharide (LPS) that is required for virulence. During serial passage in immunoincompetent hosts, such as embryonated hen's eggs and tissue culture cells, or synthetic media [21], variants emerge within the population that produce truncated LPS. Extensive passage (∼90 passages) culminates in avirulent organisms with a severely truncated, deep-rough LPS consisting of lipid A and core sugars, but no O-antigen [22][23][24].…”

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

“…First, genomic analysis identified three critical components for this complex Coxiella medium (CCM) to sustain the metabolic activity of the bacteria as well as 3 complex nutrient sources (Neopeptone, fetal bovine serum, and RPMI cell culture medium) and a high concentration of chloride and citrate buffer, mimicking the acidic environment of phagosomes (370). A transcriptomic analysis resulted in the conception of ACCM (acidified citrate cysteine medium) enriched with cysteine and Casamino Acids that support the growth of C. burnetii by Ͼ3 log 10 units after 6 days of incubation in a microaerophilic atmosphere (11,371). This medium was then optimized through the replacement of fetal bovine serum by methyl-␤-cyclodextrin and was named ACCM-2; ACCM-2 sustained 4-to 5-log 10 growth of C. burnetii over 7 days with the addition of moderate shaking (372).…”